MSACL 2023 Abstract

INTRODUCTION: Glycated hemoglobulin (HbA1c) is the gold standard for the diagnosis of diabetes mellitus and for assessing glycemic status in these patients. Although HbA1c faithfully represents the average blood glucose level over the previous 8–12 weeks, its utility in monitoring the effect of medical interventions and diabetes progression is limited. Because HbA1c is prone to fluctuations in conditions like diabetic nephropathy, anemia, and pregnancy, glycated albumin (GA) has gained traction as a potential alternative due to its shorter half-life (2–3 weeks) and higher affinity to glycation.

OBJECTIVE: To develop a fully automated high-throughput Evosep-MRM workflow for quantifying GA in plasma/serum/dried blood spots.

METHODS: Five µL of plasma was diluted in 295 µL of 50 mM Tris-formate buffer (pH 7.5) in a 96-well plate. Diluted plasma (20 µL~20 µg) was digested using a modified solvent-assisted tryptic digestion (trypsin: protein-1:40) for 60 min and quenched using 4% formic acid before drying down. The complete sample handling was performed using an Opentrons OT-2 robot. The samples were reconstituted in 300 µL 0.1% FA and 20 µL was loaded to preconditioned Evotips using the OT-2 automated loading protocol and analyzed with Evosep One coupled to TSQ Altis using the standardized 300 samples/day or 500 samples/day method. Three intense transitions of a glycated and non-glycated form of KQTALVELVK peptide were monitored. The results were reported as %glycation of albumin (%GA).

RESULTS: The assay exhibited high repeatability and intermediate precision with a median coefficient of variation <6% (intra-assay) and <10% (inter-assay) across 3 QC levels of the JCCRM 611 GA calibration standard.

The stability of the glycated peptide in plasma and serum was explored for 3 handling temperatures (-80 deg C, +40 deg C, +220 deg C) for a span of 3 days. The %GA was stable at -80 deg C and +4 deg C for 3 days, whereas GA stability at room temperature was limited to 6 hours.
Further, we investigated the correlation between Evosep-MRM %GA and Tosoh G8 HPLC %HbA1c in random patient samples. The %GA and the %HbA1C exhibited a high, positive correlation, r=0.90. Next, the optimized assay was challenged with 407 patient samples with and without gestational diabetes mellitus to assess the robustness of the assay. Data will be presented.
We also further explored dried blood spots (DBS) as a sample source for the quantification of glycated albumin. Initial extraction protocols provided efficient solubilization of the proteins and the glycated albumin was quantified with a coefficient of variation of less than 10 %.

CONCLUSION: We have successfully developed a high throughput end-to-end automated assay for the quantification of glycated albumin in plasma, serum, and dried blood spots.